System for extracting tetrodotoxin

ABSTRACT

The invention relates to a system for extracting toxins from biological tissues. The system comprises a lixiviator, an ion-exchange column, a diatomaceous silica-active carbon column, and a vacuum concentrator. Twenty to one hundred kilograms of raw material can be processed at one time when puffer fish ovaries are used as the starting material.

FIELD OF THE INVENTION

[0001] This invention relates to apparatus for extraction oftetrodotoxin. The apparatus can be used, inter alia, for batchproduction of tetrodotoxin or other toxins from animal tissues, forexample using the method described in another application of theinventors (application Ser. No. 09/695,711, filed Oct. 25, 2000,Attorney Docket No. 3519-0111P). Twenty to one hundred kilograms of rawmaterial can be processed at one time when puffer fish ovaries are used.

BACKGROUND OF THE INVENTION

[0002] A method for extraction of tetrodotoxin from animal tissues isdescribed in a prior patent application of the inventors (U.S.application Ser. No. 09/695,711, filed Oct. 25, 2000, Attorney DocketNo. 3519-0101P). That method comprises five steps as follows:

[0003] Step 1: Grind the tissues into small pieces, soak with an amountof water equal to 1.5 times by weight of the tissues and an amount of aweak organic acid, typically a carboxylic acid, preferably acetic acid,equal to 0.05%-1%, preferably 0.1%-0.3%, by weight of the tissue forseveral hours, then stir and filter quickly to obtain a lixiviatedsolution. Repeat this step 3-4 times in order to extract as much toxinas possible.

[0004] Step 2: Heat the lixiviated solution to 70-95° C. to coagulateand remove soluble proteins (“scleroprotein”).

[0005] Step 3: Adjust the pH of the lixiviated solution obtained in step1 to 6.0˜7.5 using an aqueous solution of a weak base, then put thesolution through a weakly acidic cation ion-exchange resin to enrichtetrodotoxin. Elute the bound tetrodotoxin with a weak acid.

[0006] Step 4: Adjust the pH of the obtained tetrodotoxin solution instep 3 to a range between 8 and 9 for a period of 2-4 hours, duringwhich the solution is put through a column filled with active charcoaland diatomaceous silica so as to remove inorganic salts and a fractionof the alkaline amino acids. Tetrodotoxin binds the immobile phase,which is washed with de-ionized water, then the toxin is eluted withacidic ethanol solution.

[0007] Step 5: Purify and crystallize the tetrodotoxin by concentratingthe solution obtained in step 4 under vacuum, then adjusting to analkaline pH. Vacuum dry the obtained tetrodotoxin crystals, typicallyabout 24 hours until the weight of the crystals becomes constant.

[0008] The method as described above can be run in either a batch modeor a continuous mode, but is typically run in a batch mode. Theextraction system described herein is an efficient system for continuousproduction. A production cycle can be completed within a period of 6-7days providing that the extraction is conducted 24 hours a day and20-100 kg puffer fish ovaries are loaded at one time. For a load of 20kg material, 0.8-1.2 grams tetrodotoxin can be obtained; for a load of100 kg, 4-6 grams can be obtained.

SUMMARY OF THE INVENTION

[0009] The extraction system of this invention is named IWT-112 (SeeFIG. 1). An important component of the IWT-112 system is a lixiviatingand filtering apparatus (“lixiviator”, device IWT-113). Additionalcomponents are a heater, an ion-exchange column, a diatomaceoussilica-active carbon column, a vacuum concentrator etc. The componentsof the system are arranged so that liquids flow from the outlet of onedevice into the inlet of the next device. For continuous mode operation,it is possible to arrange the downstream components so that a batch runfrom the lixiviator is directed to one of several sets of series of thedownstream components. Flow through the system can be driven either bygravity or by pressure differential established across each componentseparately or across the system as a whole or across various subsets ofthe components. For instance, a pressure differential can be establishedacross the cation-exchange column and the diatomaceous silica-activatedcharcoal column while at the same time a vacuum is drawn on thedecompression chamber of the lixiviator by an appropriate arrangement ofvalves and pressure and vacuum lines. Such a mode of operation might beutilized, for example, when starting a second extraction in thelixiviator while the chromatography steps are being completed on a firstextraction.

[0010] Lixiviator (Device IWT-113, FIG. 2):

[0011] Device IWT-113 functions to lixiviate and filter. It is made ofmetals, preferably a corrosion-resistant material like stainless steel.It comprises three dismountable parts, namely a “sealing head” at thetop, a “lixiviating barrel” in the middle and a “decompressing chamber”at the bottom.

[0012] The sealing head comprises a deceleration joint (1), a pressuregauge (2), a water inlet valve (3), a material filling inlet (4), asafety valve (14), a gas valve (15) and a degassing valve (16). Thedeceleration joint (1) transmits power to the propeller pug mill (8) byconnecting a decelerating motor at a speed of 60-120 rpm so as to enablefaster lixiviation and filtration by stirring. The propeller pug mill(8) can also help remove used raw material. The water inlet valve (3) isused to provide water for repeated lixiviation steps. After lixiviation,water can be supplied through the water inlet valve (3) to help pump outthe used raw material from the material filling inlet while stirring.

[0013] The safety valve (14) controls the pressure inside thelixiviating barrel. The pressure is typically 0.5-1.5 kg per squarecentimeters while filtering. The pressure can be elevated up to 6 kg persquare centimeters when necessary. The gas valve (15) maintains thepressure inside the lixiviating barrel by connecting to anair-compressing unit, whereas the degassing valve (16) functions torelease the pressure inside the barrel.

[0014] The sealing head (6) is removably attached to the lixiviator. Thesealing head is typically bolted on, but any removable joiner, forexample a clamping device, that can seal the sealing head against thelixiviating barrel and can tolerate the pressurization can be used. Agasket for effecting a pressure seal can be inserted between the sealinghead and the lixiviator barrel. The gasket material can be any typicalpressure gasket material, preferably an elastomeric material that isresistant to weak acid solutions.

[0015] Filtering material (9, 10) is mounted between the decompressingchamber and the lixiviator barrel. During the lixiviation process, thefiltration can be speeded by elevating the pressure in the lixiviationbarrel or reducing pressure in the decompression module. The filteringmaterial is made from filtering paper, filtering cloth and other porousfiltering material that is compatible with use between stainless steelcomponents. “Compatible with use between stainless steel components”means that the filter material will at least not promote electromotivecorrosion of the stainless steel.

[0016] The filtering material can be for example, a nylon mesh having100 to 200 meshes per square inch, a stainless steel mesh having 40 to60 meshes per square inch, or a porous metal plate having a porediameter of from 2 to 10 mm, preferably from 2 to 4 mm, more preferably2.5 to 3.5 mm. These materials can be combined in a layered arrangement.Thus, the filter material can be a combination for example, of a nylonmesh having 100 to 200 meshes per square inch, middle to high speedfiltering paper, a stainless steel mesh having 40 to 60 meshes persquare inch, and a porous metal plate having a pore diameter of 3 mm. Apreferred arrangement is to have a metal plate on the bottom, upon whichis stacked a stainless steel mesh, a filtering paper and then nylon meshon top.

[0017] The filter is preferably installed in a frame that can be removedfrom the lixivating/filtering apparatus for easy cleaning andmaintenance. Also, the filter is preferably mounted in an elastomericmaterial suitable for maintaining a pressure seal between the filter andthe walls of the lixiviator barrel and/or decompression chamber.

[0018] A vacuum valve (13) is installed on the side of the decompressionchamber, and a drain (18) at the bottom.

[0019] The lixiviator can be mounted on a stand (19).

[0020] A heater can be fitted to the lixiviator. The heater should becapable of heating the liquid to a desired temperature and maintainingthat temperature. The heater can be an oil-bath heater or steam heaterof industry standards. Alternatively, the filtered solution collectingin the decompressing chamber at the bottom of the lixiviator can bepassed to a separate heated vessel.

[0021] A second filtering apparatus is connected to the outlet of thelixiviator or of the separate heated vessel. The second filteringapparatus can be integral with the separate heated vessel. The filteringmaterial of the second filtering apparatus functions to removeprecipitate from the heated filtrate. The filtering material of thesecond filtering apparatus can be made from material similar to that ofthe filter between the lixiviator barrel and decompression chamber ofthe lixiviator. However, the filtering material of the second filteringapparatus is more preferably one having a smaller pore size, sufficientto remove small bits of precipitated protein so that such precipitatedoes not interfere with subsequent chromatographic steps. The filteringmaterial for the second filtering apparatus preferably includes a middleto high speed filtering paper, in order to separate out the smaller bitsof precipitated protein. The order of the filtering material componentsis the same as that of the first filter. Also, rather than “mesh” typefiltering material, an membrane type filter, typically made from nylonor other polymeric material, suitable for filtration driven by apressure differential is preferred as the filter material for the secondfiltration apparatus.

[0022] The second filtering apparatus is preferably operable by pressuredifferential and therefore the second filtering apparatus and theseparate heated vessel may comprise appropriate connections to pressureor vacuum lines and appropriate relief valves as needed.

[0023] Ion-exchange column (Device IWT-312):

[0024] An ion-exchange column (IWT-312) is used to remove proteins,peptides and their derivatives from the tetrodotoxin solution duringenrichment and purification of tetrodotoxin. If not removed, thesesubstances produce a high viscosity solution and also interfere withprecipitation of the tetrodotoxin later in processing.

[0025] Diatomaceous Silica-active Charcoal Column (Device IWT-412):

[0026] A column comprising diatomaceous silica and activated charcoal isused to remove basic amino acids remaining in the eluate from theion-exchange column.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027]FIG. 1—Flow chart of IWT-112 TTX Extraction System.

[0028]FIG. 2—Lixiviator—Apparatus IWT-113.

[0029]FIG. 3—HPLC profile of TTX obtained in Example 1.

[0030]FIG. 4—HPLC profile of TTX obtained in Example 2.

DETAILED DESCRIPTION OF THE INVENTION

[0031] The extraction procedure using the system of this inventioncomprises:

[0032] 1. Lixiviation with Water

[0033] 20-100 kg toxic tissues, for example ovaries of puffer fish, arecrushed into pieces less than 1 cc³, and put into a IWT-113 lixiviator(See FIG. 2). The lixiviator is filled with de-ionized water to 1.5times the weight of the ovaries and acetic acid is added to 0.1 to 1% byweight of the ovaries. The propeller pug mill is run to stir the mixturefor a few hours at room temperature. The vacuum pump is run todecompress the decompression chamber and begin filtering for severalminutes. Then the compressor is run to adjust the pressure at the upperpart of the lixiviator to 0.5 to1 kg/cm². The filtrate is collected,then heated quickly to 70-90° C. for 3-25 minutes. The filtrate iscooled and again filtered to remove the precipitate. The clarifiedfiltrate is a yellow transparent liquid, called “first clear lixiviatedliquid”.

[0034] The lixiviation is repeated as above and the second lixiviatedclear liquid is collected. Repeat the lixiviation process to obtain thethird and the fourth lixiviated liquids. The fourth lixiviated liquid iscollected only when extremely or highly toxic tissues are used. Formildly toxic tissues, only the third lixiviated liquid is obtained.

[0035] The first, second, third (and fourth) lixiviated liquids arepooled, and the toxin content of the liquid is determined by bioassay orhigh performance liquid chromatography (HPLC). See, e.g. Chinese patentapplication 00132673.2, filed Nov. 22, 2000, or U.S. application Ser.No. ______, filed ______, Attorney Docket No. 3519-0110P.

[0036] 2. Ion-Exchange Enrichment and TTX-Separation Process

[0037] The pH of the pooled clear lixiviated liquid is adjusted to6.0˜7.5, preferably using a strong amine base, typically ammonia. Anyprecipitate that forms should be removed, e.g by filtration. The clearlixiviated liquid is then put through a cation-exchange resin column(Device IWT-312). A preferred resin is a weakly acidic ammonium cationion-exchange resin, such as D-152 (Nankai University Chemical Factory,Tianjing, P.R. China.) A typical flow rate for loading the column is2000 to 3750 mL/hr. Thin layer chromatography (TLC) on a silica plate isused to monitor whether or not toxin leaks from the outflow. TLC can beperformed on a silica gel thin-layer plate eluted with n-butanol: aceticacid: water (2:1:1). TTX can be detected by spraying the plate with 10%KOH solution after it is eluted, baking for 10 minutes at 110° C., andobserving under a 365 nm ultraviolet lamp. TTX is detected as bluefluorescent spots with R_(f)=0.38−0.40. If toxin leaks, the resin columnshould be replaced with a new one immediately.

[0038] After all the clear lixiviated liquid passes through the column,the column is washed with de-ionized water until there is no proteinpresent in the outflow. The outflow can be monitored for protein contentby any typical method in the art, for example by measuring absorbance of280 nm light. The column is then eluted with a solution of 5 to 12% (byvolume) of a weak organic acid, preferably acetic acid, in water,typically at a flow rate of 500 to 2500 mL/hr. The eluate is collectedin fractions, preferably of about 1500 mL per portion, and each fractionis assayed for TTX content by thin layer chromatography (TLC).

[0039] 3. Purification with Active Charcoal

[0040] The eluate from the ion-exchange column that contain TTX arepooled and the pH of the solution is adjusted to 8 to 9 with a strongbase, preferably aqueous ammonia. The pH is maintained for a period of 2to 4 hours, within which time the solution is passed through a columnthat is packed with active charcoal and diatomaceous silica. Thediatomaceous silica-active carbon column comprises two layers, with theupper layer being diatomaceous silica and the lower layer being activecharcoal. The layer of diatomaceous silica has a {fraction (1/10)} to ½,preferably {fraction (1/10)}-⅕, the thickness of the layer of activecharcoal.

[0041] The column is washed with de-ionized water, then eluted with anethanol solution that contains acid (the concentration of ethanol isbetween 0.5% to 40%, preferably 15% to 30% by volume; the concentrationof the acid is from and 0.2 to 0.6% by volume. The acid is preferably aweak organic acid, typically acetic acid. Elution of TTX from the columncan be monitored by TLC.

[0042] Concentration and Crystallization

[0043] The eluate from the silica-charcoal column is concentrated byvacuum evaporation of the solvent, preferably to about {fraction(1/10)}th the volume, Or about 0.5-1 mL per kilogram raw material used.The concentrated solution is cooled to 18-25° C. (room temperature) andthe pH is adjusted 8 to 9. A strong amine base, preferably ammonia, isused to adjust the pH. The solution is left standing to precipitate TTX.The crystalline TTX is collected, preferably by filtration and thenwashed with de-ionized water several times. The crystalline TTX isdried, for example by vacuum evaporation, and redissolved in a dilutesolution of an organic acid, preferably acetic acid. The acid solutionhas a concentration of about 3-7%, preferably 5%, by volume of the acid.The pH is again adjusted to 8 to 9 as above and the TTX isreprecipitated, washed and dried as above. The recrystallization can berepeated, e.g. another 1 or 2 times. The TTX crystals obtained are driedto constant weight and the purity of the produc is determined by HPLC.

EXAMPLES

[0044] The following examples serve to illustrate the invention, but arein no way intended to limit the invention.

EXAMPLE 1

[0045] Twenty kilograms ovaries of puffer fish were ground into piecesless than 1 cc³, and put into an IWT-113 lixiviator. Thirty litersde-ionized water and 30 milliliters acetic acid were added. Thepropeller pug mill was started to stir the mixture for 10 hours at roomtemperature. Then a vacuum pump was run to enable the filtrate startflowing out from the lixiviator, followed by turning on an aircompressor to speed the filtration. After filtering, another 30 litersde-ionized water was added to repeat the lixiviation process. Thelixiviation process was repeated totally four times.

[0046] The filtrates were merged, and then heated at 80° C. for 5minutes. After cooling down, the filtrates were sent into a secondIWT-113 lixiviator to separate out the precipitated scleroprotein andobtain clear liquid. The pH of the clear liquid was adjusted to 7.5using aqueous ammonia. Then the solution was put through an ion-exchangecolumn (device IWT-312) which was a NH₄ ⁺ weakly acidic cationion-exchange resin (D-152, (Nankai University Chemical Factory,Tianjing, P.R. China.) column having a diameter of 6 cm and a height of1 meter, filled with resin by Nankai University Chemical Factory,Tianjing, China. After the solution all passed through, the column waswashed clean with de-ionized water, then was eluted with 10% aqueousacetic acid solution to get a TTX eluate. Subsequently, strong aqueousammonia was used to adjust the pH of the TTX eluate to 8.5. Theresulting solution was put through a diatomaceous silica-active charcoalcolumn (device IWT-412), in which a diatomaceous silica layer was placedabove an active charcoal layer, and the diatomaceous silica layer was{fraction (1/15)} the thickness of the active charcoal layer. The TTXwas adsorbed on the active charcoal. Then a 20% aqueous ethanol solutioncontaining 0.2% acetic acid was used to elute TTX, and the eluate wasput into a rotating evaporator to concentrate to about 15 milliliters.After cooling to 22° C., the pH of the concentrated solution wasadjusted with concentrated ammonia to 9, and the solution was put into arefrigerator to be cooled further and to crystallize the TTX. Thecrystals were separated out and dissolved in 5% acetic acid, then the pHof the resulting solution was adjusted with strong ammonia to 9, and TTXin the solution was crystallized and precipitated again. Next, the TTXcrystals were placed in a vacuum drier and were dried for 24 hours sothat their weight became constant. The TTX content of the dried productwas determined by high performance liquid chromatograph (HPLC) to be85.4% (See FIG. 3). From 20 kilograms ovaries, 1210 milligrams TTXcrystals were obtained, an equivalent of 6.05 grams per 100 kilogramsovaries.

EXAMPLE 2

[0047] One hundred kilograms puffer fish ovaries were ground, and 150 Lwater and 150 mL 0.5% acetic acid were added. The tissue was lixiviatedand the remaining process steps were performed as in Example 1. 5.82 gTTX crystals of 80.0% purity by HPLC were obtained (See FIG. 4).

We claim:
 1. A system for extracting compounds from biological tissuescomprising: a) a lixiviating and filtering apparatus, comprising asealing head at the top, a lixiviating barrel in the middle and adecompressing chamber at the bottom, wherein the sealing head comprisesa gas inlet valve connecting to a pressurizing source and a degassingvalve, and the decompressing chamber comprises a connection to a vacuumsource; wherein a filtering material compatible with stainless steelcomponents and capable of sustaining a pressure differential of from0.5-6.0 kg per square centimeters, preferably 0.5-1.5 kg per squarecentimeters without rupturing is mounted between the decompressingchamber and the lixiviator barrel; b) a heater that is fitted to thelixiviating and filtering apparatus or is alternatively a separateheated vessel connected to the decompressing chamber of the lixiviatingand filtering apparatus; c) a second filtration apparatus connected toan outflow from the lixiviating and filtering apparatus or alternativelyto the outflow of the separately heated vessel; d) a cation exchangecolumn connected to the outflow of the second filtration apparatus; e) adiatomaceous silica-active carbon column connected to the outflow of thecation exchange column and comprising two layers, an upper layer ofdiatomaceous silica and a lower layer of active charcoal; f) anapparatus for vacuum evaporation of a solvent connected to the outflowof the diatomaceous silica-active carbon column.
 2. The system of claim1, wherein the sealing head of the lixiviating and filtering apparatuscomprises a deceleration joint, a pressure gauge, a water inlet valve, amaterial filling inlet, and a safety valve.
 3. The system of claim 1 orclaim 2, wherein the lixiviating barrel of the lixiviating and filteringapparatus comprises a propeller pug mill and a deceleration joint thattransmits power to the propeller pug mill by connecting a deceleratingmotor.
 4. The system of claim 1 wherein the filtering material of thelixiviating and filtering apparatus comprises one or more of nylonfiltering mesh, stainless steel mesh, filtering paper and a porous metalplate.
 5. The system of claim 1, wherein the filtering material of thelixiviating and filtering apparatus is a combination of a nylonfiltering mesh, a stainless steel mesh, a filtering paper and a porousmetal plate.
 6. The system of claim 5, wherein the nylon filtering meshhas 100-200 meshes per square inch, the filter paper is a middle-highspeed filtering paper, the stainless steel mesh has 20-100 meshes persquare inch, preferably 40-60 meshes per square inch, and the porousmetal plate has a pore diameter of 2.5 to 3.5 mm.
 7. The system of claim1, wherein in the diatomaceous silica-active carbon column, the layer ofdiatomaceous silica has a thickness {fraction (1/10)} to ½, preferably{fraction (1/10)} to ⅕, of the thickness of the layer of activecharcoal.
 8. A method for extracting tetrodotoxin from biologicaltissues comprising extracting the tetrodotoxin from the tissues usingthe system of claim 1.